The goal of this project is the identification of nonlethal mutations in the DEN virus genome which when incorporated into the infectious cDNA clone (project# Z01 BK 07001-02) will reduce virulence. (1) Nuclear localization of the dengue virus capsid (C) protein. We detected the presence of a nuclear localization signal (NLS) in the carboxy-terminal 40 amino acids (aa) of the DEN4 C. Substitution mutations were created within suspected NLSs in the 40 aa domain, and their nuclear transport was assessed. Only a mutant in which all (4) possible NLSs had been eliminated appeared to be altered in nuclear transport, suggesting nucleus-dependent steps in the replication of flaviviruses which can be tested by incorporation of selected mutations into infectious cDNA. (2) Ubiquitination of the dengue virus C. This work was completed. Membrane integration of C appeared to stabilize the molecule against ubiquitin- dependent degradation (manuscript in prep). (3) Membrane integration of C. The flavivirus C contains a conserved internal 20-aa hydrophobic domain. We demonstrated that this domain functions as a signal-anchor (SA) to mediate the membrane integration of C. Function of this SA is essential for efficient recognition of the prM signal peptide, which lies immediately downstream from C in the flavivirus polyprotein. (4) Effect of mutations in the DEN NS2B protein on protease activity of NS3. To further characterize the requirement of NS2B for the protease activity of the virus-coded protease, NS3, we mutagenized a 40-aa domain in NS2B indispensable for protease activity. >60 mutants were isolated, and autocleavage at the NS2B-NS3 site was analyzed in vivo and in vitro. Three of 5 aas conserved among flaviviruses in the 40-aa domain of NS2B were absolutely required for cleavage. Three additional semi-conserved aas were also required. Some mutations resulted in partial defects in cleavage, thus defining potential sites for the introduction of attenuating mutations into the dengue genome (manuscript in prep). (5) Cleavage of the NS1-NS2A site. This cleavage is known to occur after a conserved motif which is a consensus site for cleavage by the ER resident protease, signal peptidase (SP). However, the motif does not occur in the context of a signal peptide, a requirement for recognition of the motif by SP. Cleavage requires the carboxy-terminal 8 aa of NS1 and the amino-terminal 70%of NS2A. This year, we demonstrated that cleavage takes place in vitro in the presence of microsomes containing ER and its resident proteases. ER depleted of soluble intralumenal enzymes was still active for cleavage, suggesting that the cleavage enzyme is ER membrane-bound. The hypothesis that the NS1-NS2A entity is by itself an autoprotease was investigated by selectively mutagenizing all aas that could possibly be required at the active site of known types of proteases. Results suggest that NS1-NS2A is not an autoprotease and that NS1-NS2A cleavage is catalyzed by SP in a novel fashion.